Soybean variety d4923560

ABSTRACT

The invention relates to the soybean variety designated D4923560. Provided by the invention are the seeds, plants and derivatives of the soybean variety D4923560. Also provided by the invention are tissue cultures of the soybean variety D4923560 and the plants regenerated therefrom. Still further provided by the invention are methods for producing soybean plants by crossing the soybean variety D4923560 with itself or another soybean variety and plants produced by such methods.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of soybeanbreeding. In particular, the invention relates to the novel soybeanvariety D4923560.

2. Description of Related Art

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The goal is to combine in a single variety animproved combination of desirable traits from the parental germplasm.These important traits may include higher seed yield, resistance todiseases and insects, better stems and roots, tolerance to drought andheat, better agronomic quality, resistance to herbicides, andimprovements in compositional traits.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of variety used commercially (e.g., F₁ hybrid variety, purelinevariety, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection andbackcrossing.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable variety. This approach hasbeen used extensively for breeding disease-resistant varieties (Bowerset al., 1992; Nickell and Bernard, 1992). Various recurrent selectiontechniques are used to improve quantitatively inherited traitscontrolled by numerous genes. The use of recurrent selection inself-pollinating crops depends on the ease of pollination, the frequencyof successful hybrids from each pollination, and the number of hybridoffspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulvarieties produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for generally three or more years. The best lines arecandidates for new commercial varieties. Those still deficient in a fewtraits may be used as parents to produce new populations for furtherselection.

These processes, which lead to the final step of marketing anddistribution, may take as much as eight to 12 years from the time thefirst cross is made. Therefore, development of new varieties is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to one or more widely grownstandard varieties. Single observations are generally inconclusive,while replicated observations provide a better estimate of geneticworth.

The goal of plant breeding is to develop new, unique and superiorsoybean varieties and hybrids. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. Each year, the plant breederselects the germplasm to advance to the next generation. This germplasmis grown under unique and different geographical, climatic and soilconditions, and further selections are then made, during and at the endof the growing season. The varieties which are developed areunpredictable. This unpredictability is because the breeder's selectionoccurs in unique environments, with no control at the DNA level (usingconventional breeding procedures), and with millions of differentpossible genetic combinations being generated. A breeder of ordinaryskill in the art cannot predict the final resulting lines he develops,except possibly in a very gross and general fashion. The same breedercannot produce the same variety twice by using the exact same originalparents and the same selection techniques. This unpredictability resultsin the expenditure of large amounts of research monies to developsuperior new soybean varieties.

The development of new soybean varieties requires the development andselection of soybean varieties, the crossing of these varieties andselection of progeny from the superior hybrid crosses. The hybrid seedis produced by manual crosses between selected male-fertile parents orby using male sterility systems. Hybrids may be identified by usingcertain single locus traits such as pod color, flower color, pubescencecolor or herbicide resistance which indicate that the seed is truly ahybrid. Additional data on parental lines as well as the phenotype ofthe hybrid influence the breeder's decision whether to continue with thespecific hybrid cross.

Pedigree breeding and recurrent selection breeding methods are used todevelop varieties from breeding populations. Breeding programs combinedesirable traits from two or more varieties or various broad-basedsources into breeding pools from which varieties are developed byselfing and selection of desired phenotypes. The new varieties areevaluated to determine which have commercial potential.

Pedigree breeding is commonly used for the improvement ofself-pollinating crops. Two parents which possess favorable,complementary traits are crossed to produce an F₁. An F₂ population isproduced by selfing one or several F₁'s. Selection of the bestindividuals may begin in the F₂ population (or later depending upon thebreeders objectives); then, beginning in the F₃, the best individuals inthe best families can be selected. Replicated testing of families canbegin in the F₃ or F₄ generation to improve the effectiveness ofselection for traits with low heritability. At an advanced stage ofinbreeding (i.e., F₆ and F₇), the best lines or mixtures ofphenotypically similar lines are tested for potential release as newvarieties.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genetic loci for simplyinherited, highly heritable traits into a desirable homozygous varietywhich is the recurrent parent. The source of the trait to be transferredis called the donor or nonrecurrent parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., variety)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., variety) and the desirable trait transferredfrom the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, soybean breeders commonly harvest one ormore pods from each plant in a population and thresh them together toform a bulk. Part of the bulk is used to plant the next generation andpart is put in reserve. The procedure has been referred to as modifiedsingle-seed descent or the pod-bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to thresh pods with a machine than to remove oneseed from each by hand for the single-seed procedure. The multiple-seedprocedure also makes it possible to plant the same number of seeds of apopulation each generation of inbreeding. Enough seeds are harvested tomake up for those plants that did not germinate or produce seed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987a,b).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current varieties. In addition toshowing superior performance, there must be a demand for a new varietythat is compatible with industry standards or which creates a newmarket. The introduction of a new variety will incur additional costs tothe seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new variety should take into consideration research and developmentcosts as well as technical superiority of the final variety. Forseed-propagated varieties, it must be feasible to produce seed easilyand economically.

Soybean, Glycine max (L), is an important and valuable field crop. Thus,a continuing goal of plant breeders is to develop stable, high yieldingsoybean varieties that are agronomically sound. The reasons for thisgoal are to maximize the amount of grain produced on the land used andto supply food for both animals and humans. To accomplish this goal, thesoybean breeder must select and develop soybean plants that have thetraits that result in superior varieties.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to seed of the soybeanvariety D4923560. The invention also relates to plants produced bygrowing the seed of the soybean variety D4923560, as well as thederivatives of such plants. As used herein, the term “plant” includesplant cells, plant protoplasts, plant cells of a tissue culture fromwhich soybean plants can be regenerated, plant calli, plant clumps, andplant cells that are intact in plants or parts of plants, such aspollen, flowers, seeds, pods, leaves, stems, and the like.

Another aspect of the invention relates to a tissue culture ofregenerable cells of the soybean variety D4923560, as well as plantsregenerated therefrom, wherein the regenerated soybean plant is capableof expressing all the physiological and morphological characteristics ofa plant grown from the soybean seed designated D4923560.

Yet another aspect of the current invention is a soybean plantcomprising a single locus conversion of the soybean variety D4923560,wherein the soybean plant is otherwise capable of expressing all thephysiological and morphological characteristics of the soybean varietyD4923560. In particular embodiments of the invention, the single locusconversion may comprise a transgenic gene which has been introduced bygenetic transformation into the soybean variety D4923560 or a progenitorthereof. In still other embodiments of the invention, the single locusconversion may comprise a dominant or recessive allele. The locusconversion may confer potentially any trait upon the single locusconverted plant, including herbicide resistance, insect resistance,resistance to bacterial, fungal, or viral disease, male fertility orsterility, and improved nutritional quality.

Still yet another aspect of the invention relates to a first generation(F₁) hybrid soybean seed produced by crossing a plant of the soybeanvariety D4923560 to a second soybean plant. Also included in theinvention are the F₁ hybrid soybean plants grown from the hybrid seedproduced by crossing the soybean variety D4923560 to a second soybeanplant. Still further included in the invention are the seeds of an F₁hybrid plant produced with the soybean variety D4923560 as one parent,the second generation (F₂) hybrid soybean plant grown from the seed ofthe F₁ hybrid plant, and the seeds of the F₂ hybrid plant.

Still yet another aspect of the invention is a method of producingsoybean seeds comprising crossing a plant of the soybean varietyD4923560 to any second soybean plant, including itself or another plantof the variety D4923560. In particular embodiments of the invention, themethod of crossing comprises the steps of a) planting seeds of thesoybean variety D4923560; b) cultivating soybean plants resulting fromsaid seeds until said plants bear flowers; c) allowing fertilization ofthe flowers of said plants; and, d) harvesting seeds produced from saidplants.

Still yet another aspect of the invention is a method of producinghybrid soybean seeds comprising crossing the soybean variety D4923560 toa second, distinct soybean plant which is nonisogenic to the soybeanvariety D4923560. In particular embodiments of the invention, thecrossing comprises the steps of a) planting seeds of soybean varietyD4923560 and a second, distinct soybean plant, b) cultivating thesoybean plants grown from the seeds until the plants bear flowers; c)cross pollinating a flower on one of the two plants with the pollen ofthe other plant, and d) harvesting the seeds resulting from the crosspollinating.

Still yet another aspect of the invention is a method for developing asoybean plant in a soybean breeding program comprising: obtaining asoybean plant, or its parts, of the variety D4923560; and b) employingsaid plant or parts as a source of breeding material using plantbreeding techniques. In the method, the plant breeding techniques may beselected from the group consisting of recurrent selection, massselection, bulk selection, backcrossing, pedigree breeding, geneticmarker-assisted selection and genetic transformation. In certainembodiments of the invention, the soybean plant of variety D4923560 isused as the male or female parent.

Still yet another aspect of the invention is a method of producing asoybean plant derived from the soybean variety D4923560, the methodcomprising the steps of: (a) preparing a progeny plant derived fromsoybean variety D4923560 by crossing a plant of the soybean varietyD4923560 with a second soybean plant, wherein a sample of the seed ofthe soybean variety D4923560 was deposited under ATCC Accession No.______; and (b) crossing the progeny plant with itself or a second plantto produce a progeny plant of a subsequent generation which is derivedfrom a plant of the soybean variety D4923560. In one embodiment of theinvention, the method further comprises: (c) crossing the progeny plantof a subsequent generation with itself or a second plant; and (d)repeating steps (b) and (c) for at least 2-10 additional generations toproduce an inbred soybean plant derived from the soybean varietyD4923560. Also provided by the invention is a plant produced by this andthe other methods of the invention. Plant variety D4923560-derivedplants produced by this and the other methods of the invention describedherein may, in certain embodiments of the invention, be further definedas comprising at least two, including at least three, four, six, eightand twelve of the traits of plant variety D4923560 given in Table 1.

In another embodiment of the invention, the method of producing asoybean plant derived from the soybean variety D4923560 furthercomprises: (a) crossing the soybean variety D4923560-derived soybeanplant with itself or another soybean plant to yield additional soybeanvariety D4923560-derived progeny soybean seed; (b) growing the progenysoybean seed of step (a) under plant growth conditions, to yieldadditional soybean variety D4923560-derived soybean plants; and (c)repeating the crossing and growing steps of (a) and (b) from 0 to 7times to generate further soybean variety D4923560-derived soybeanplants. The invention still further provides a soybean plant produced bythis and the foregoing methods.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention provides methods and composition relating toplants, seeds and derivatives of the soybean variety D4923560. Soybeanvariety D4923560 is adapted to the upper Midwest of the United States.Soybean variety D4923560 was developed from an initial cross ofAG1602/DRM2201A0R. The breeding history can be summarized as follows:

Generation Year Location Description Cross July 2001 RMBS F₁ October2001 KEHI Bulk F₂ February 2002 KEHI Bulk F₃ May 2002 RMBS Bulk F₄ May2003 RMBS Progeny Row Yield Trial

Yield Testing:

Generation Year No. of Locations Rank No. of Entries F₅ 2004 3 4 50 F₆2005 11 3 50 F₇ 2006 20 3 40

The soybean variety D4923560 has been judged to be uniform for breedingpurposes and testing. The variety D4923560 can be reproduced by plantingand growing seeds of the variety under self-pollinating orsib-pollinating conditions, as is known to those of skill in theagricultural arts. Variety D4923560 shows no variants other than whatwould normally be expected due to environment or that would occur foralmost any characteristic during the course of repeated sexualreproduction. The results of an objective description of the variety arepresented below, in Table 1. Those of skill in the art will recognizethat these are typical values that may vary due to environment and thatother values that are substantially equivalent are within the scope ofthe invention.

TABLE 1 Phenotypic Description of Variety D4923560 Trait PhenotypeRelative Maturity 1.5 Round-up Ready Resistant STS Susceptible FlowerPurple Pubescence Brown Hilum Black Pod Color Brown Hypocotyl ColorLight purple Seed Shape Spherical flattened Seed Coat Color Yellow LeafShape Ovate Growth Habit Indeterminate Resistance Phytophthora root rotallele Rps1k SCN Race 3 Susceptible SCN Race 14 Susceptible Root KnotNematode Susceptible

The performance characteristics of soybean variety D4923560 were alsoanalyzed and comparisons were made with competing varieties. The resultsof the analysis are presented below, in Table 2.

TABLE 2 Performance Comparison of Variety D4923560 Versus CompetingVarieties PRODUCT NAME YLD BE YLD SELIN MAT PRM PHT EMR SDV LDG PSC MYLDIDE BRR BRS SCL D4923560 50.85 50.75 102.1 19.54 1.6 31.1 2.6 2.67 21.79 105.06 3.83 8 2 1.33 S14-K6 49.64 49.63 101.4 18.88 1.48 32.2 2.52.83 2 2.36 102.44 6.28 1 9 2 AG1102 48.52 48.82 101.6 17.75 1.3 31.92.5 2.67 2.17 2.21 100.31 3.55 0 10 2 AG1401 48.27 48.15 99.33 18 1.3234.3 2.9 2.67 2.33 2.07 97.06 4.23 2 7 1 S14-A7 47.99 48.03 98.03 211.74 32.8 3 2.67 2.17 2.57 98.22 3.74 0 10 1.33 AG1402 47.38 47.14 99.8717.96 1.35 29.6 3 2.83 2.17 2.5 104.83 4.65 2 7 1.67 91M60 46.99 47.0198.88 18.75 1.35 33.4 3.1 2.5 3.17 2.64 99.64 4 2 8 2 S11-L2 46.69 46.62101.5 15.08 1.03 35 2.8 2.33 2 4 96.13 4.18 1 9 1.33 AG1002 46.61 46.3999.22 16.67 1.12 31.9 3.5 3 2.17 3.36 93.33 4.25 0 10 1.33 DKB10-5246.61 46.69 100.3 16.08 1.07 33.8 2.9 2.83 2.17 2.79 101.63 4.68 3 61.67 S12-B9 46.28 46.1 100.7 15.88 0.93 31.9 3.1 2.67 1.67 3.36 99.115.42 2 8 1.33 91M13 45.09 45.24 99.14 14.75 1.01 30.4 3 2.33 1.67 3.1494.69 4.32 6 3 2 1212RR/N 43.9 44.15 96.61 18.04 1.43 30.8 3.1 2.67 23.07 93.02 4 6 3 1.33 S13-Y3 43.34 43.6 95.9 18.25 1.37 33.1 3.3 3 3.173.86 89.72 4.63 8 1 1.33 Check Means 47.69 47.63 100 17.59 1.27 31.932.98 2.79 2.21 2.54 98.88 4.17 1 8.5 1.5 Experiment 47.8 47.79 100.1 181.34 32.74 2.93 2.7 2.2 2.82 100 4.26 3.38 6.03 1.77 Means No. Of Tests20 20 12 12 9 5 5 3 3 7 4 5 1 1 1 Legend Abbreviations BRI = Brown StemRot CAN = Canopy EMR = Emergence IDE = Iron Chlorosis (Early) IDL = IronChlorosis (Late) LDG = Lodging LLN = Linolenic Acid MAT = Maturity MST =Moisture OIL = Oil YLD = Yield PHT = Plant Height PRO = Protein HWT=Harvested Plot Weight PRM = Predicted Relative Maturity PSC =Phenotypic Score PSE = Early Phenotypic Rating SELIN = Selection IndexSCL = White mold-field rating SCN = Soybean Cyst Nematode SDS = SuddenDeath Syndrome SDV = Seedling Vigor Rating SWT = Seed Weight YLD_BE =Yield Best Estimate YLD COUNT = Yield Count (No. Evaluated Plots) MYLD =Mean Yield BRR = Resistant Brown Stem Rot BRS = Susceptible Brown StemRot

I. BREEDING SOYBEAN VARIETY D4923560

One aspect of the current invention concerns methods for crossing thesoybean variety D4923560 with itself or a second plant and the seeds andplants produced by such methods. These methods can be used forpropagation of the soybean variety D4923560, or can be used to producehybrid soybean seeds and the plants grown therefrom. Hybrid soybeanplants can be used by farmers in the commercial production of soyproducts or may be advanced in certain breeding protocols for theproduction of novel soybean varieties. A hybrid plant can also be usedas a recurrent parent at any given stage in a backcrossing protocolduring the production of a single locus conversion of the soybeanvariety D4923560.

The variety of the present invention is well suited to the developmentof new varieties based on the elite nature of the genetic background ofthe variety. In selecting a second plant to cross with D4923560 for thepurpose of developing novel soybean varieties, it will typically bedesired to choose those plants which either themselves exhibit one ormore selected desirable characteristics or which exhibit the desiredcharacteristic(s) when in hybrid combination. Examples of potentiallydesired characteristics include seed yield, lodging resistance,emergence, seedling vigor, disease tolerance, maturity, plant height,high oil content, high protein content and shattering resistance.

Any time the soybean variety D4923560 is crossed with another,different, variety, first generation (F₁) soybean progeny are produced.The hybrid progeny are produced regardless of characteristics of the twovarieties produced. As such, an F₁ hybrid soybean plant may be producedby crossing D4923560 with any second soybean plant. The second soybeanplant may be genetically homogeneous (e.g., inbred) or may itself be ahybrid. Therefore, any F₁ hybrid soybean plant produced by crossingsoybean variety D4923560 with a second soybean plant is a part of thepresent invention.

Soybean plants (Glycine max L.) can be crossed by either natural ormechanical techniques (see, e.g., Fehr, 1980). Natural pollinationoccurs in soybeans either by self pollination or natural crosspollination, which typically is aided by pollinating organisms. Ineither natural or artificial crosses, flowering and flowering time arean important consideration. Soybean is a short-day plant, but there isconsiderable genetic variation for sensitivity to photoperiod (Hamner,1969; Criswell and Hume, 1972). The critical day length for floweringranges from about 13 h for genotypes adapted to tropical latitudes to 24h for photoperiod-insensitive genotypes grown at higher latitudes(Shibles et al., 1975). Soybeans seem to be insensitive to day lengthfor 9 days after emergence. Photoperiods shorter than the critical daylength are required for 7 to 26 days to complete flower induction(Borthwick and Parker, 1938; Shanmugasundaram and Tsou, 1978).

Sensitivity to day length is an important consideration when genotypesare grown outside of their area of adaptation. When genotypes adapted totropical latitudes are grown in the field at higher latitudes, they maynot mature before frost occurs. Plants can be induced to flower andmature earlier by creating artificially short days or by grafting (Fehr,1980). Soybeans frequently are grown in winter nurseries located at sealevel in tropical latitudes where day lengths are much shorter thantheir critical photoperiod. The short day lengths and warm temperaturesencourage early flowering and seed maturation, and genotypes can producea seed crop in 90 days or fewer after planting. Early flowering isuseful for generation advance when only a few self-pollinated seeds perplant are needed, but not for artificial hybridization because theflowers self-pollinate before they are large enough to manipulate forhybridization. Artificial lighting can be used to extend the natural daylength to about 14.5 h to obtain flowers suitable for hybridization andto increase yields of self-pollinated seed.

The effect of a short photoperiod on flowering and seed yield can bepartly offset by altitude, probably due to the effects of cooltemperature (Major et al., 1975). At tropical latitudes, varietiesadapted to the northern U.S. perform more like those adapted to thesouthern U.S. at high altitudes than they do at sea level.

The light level required to delay flowering is dependent on the qualityof light emitted from the source and the genotype being grown. Bluelight with a wavelength of about 480 nm requires more than 30 times theenergy to inhibit flowering as red light with a wavelength of about 640nm (Parker et al., 1946).

Temperature can also play a significant role in the flowering anddevelopment of soybean (Major et al., 1975). It can influence the timeof flowering and suitability of flowers for hybridization. Temperaturesbelow 21° C. or above 32° C. can reduce floral initiation or seed set(Hamner, 1969; van Schaik and Probst, 1958). Artificial hybridization ismost successful between 26° C. and 32° C. because cooler temperaturesreduce pollen shed and result in flowers that self-pollinate before theyare large enough to manipulate. Warmer temperatures frequently areassociated with increased flower abortion caused by moisture stress;however, successful crosses are possible at about 35° C. if soilmoisture is adequate.

Soybeans have been classified as indeterminate, semi-determinate, anddeterminate based on the abruptness of stem termination after floweringbegins (Bernard and Weiss, 1973). When grown at their latitude ofadaptation, indeterminate genotypes flower when about one-half of thenodes on the main stem have developed. They have short racemes with fewflowers, and their terminal node has only a few flowers.Semi-determinate genotypes also flower when about one-half of the nodeson the main stem have developed, but node development and flowering onthe main stem stops more abruptly than on indeterminates. Their racemesare short and have few flowers, except for the terminal one, which mayhave several times more flowers than those lower on the plant.Determinate varieties begin flowering when all or most of the nodes onthe main stem have developed. They usually have elongated racemes thatmay be several centimeters in length and may have a large number offlowers. Stem termination and flowering habit are reported to becontrolled by two major genes (Bernard and Weiss, 1973).

Soybean flowers typically are self-pollinated on the day the corollaopens. The amount of natural crossing, which is typically associatedwith insect vectors such as honeybees, is approximately 1% for adjacentplants within a row and 0.5% between plants in adjacent rows. Thestructure of soybean flowers is similar to that of other legume speciesand consists of a calyx with five sepals, a corolla with five petals, 10stamens, and a pistil (Carlson, 1973). The calyx encloses the corollauntil the day before anthesis. The corolla emerges and unfolds to exposea standard, two wing petals, and two keel petals. An open flower isabout 7 mm long from the base of the calyx to the tip of the standardand 6 mm wide across the standard. The pistil consists of a single ovarythat contains one to five ovules, a style that curves toward thestandard, and a club-shaped stigma. The stigma is receptive to pollenabout 1 day before anthesis and remains receptive for 2 days afteranthesis, if the flower petals are not removed. Filaments of ninestamens are fused, and the one nearest the standard is free. The stamensform a ring below the stigma until about 1 day before anthesis, thentheir filaments begin to elongate rapidly and elevate the anthers aroundthe stigma. The anthers dehisce on the day of anthesis, pollen grainsfall on the stigma, and within 10 h the pollen tubes reach the ovary andfertilization is completed (Johnson and Bernard, 1963).

Self-pollination occurs naturally in soybean with no manipulation of theflowers. For the crossing of two soybean plants, it is typicallypreferable, although not required, to utilize artificial hybridization.In artificial hybridization, the flower used as a female in a cross ismanually cross pollinated prior to maturation of pollen from the flower,thereby preventing self fertilization, or alternatively, the male partsof the flower are emasculated using a technique known in the art.Techniques for emasculating the male parts of a soybean flower include,for example, physical removal of the male parts, use of a genetic factorconferring male sterility, and application of a chemical gametocide tothe male parts.

For artificial hybridization employing emasculation, flowers that areexpected to open the following day are selected on the female parent.The buds are swollen and the corolla is just visible through the calyxor has begun to emerge. Usually no more than two buds on a parent plantare prepared, and all self-pollinated flowers or immature buds areremoved with forceps. Special care is required to remove immature budsthat are hidden under the stipules at the leaf axil, and could developinto flowers at a later date. The flower is grasped between the thumband index finger and the location of the stigma determined by examiningthe sepals. A long, curvy sepal covers the keel, and the stigma is onthe opposite side of the flower. The calyx is removed by grasping asepal with the forceps, pulling it down and around the flower, andrepeating the procedure until the five sepals are removed. The exposedcorolla is removed by grasping it just above the calyx scar, thenlifting and wiggling the forceps simultaneously. Care is taken to graspthe corolla low enough to remove the keel petals without injuring thestigma. The ring of anthers is visible after the corolla is removed,unless the anthers were removed with the petals. Cross-pollination canthen be carried out using, for example, petri dishes or envelopes inwhich male flowers have been collected. Desiccators containing calciumchloride crystals are used in some environments to dry male flowers toobtain adequate pollen shed.

It has been demonstrated that emasculation is unnecessary to preventself-pollination (Walker et al., 1979). When emasculation is not used,the anthers near the stigma frequently are removed to make it clearlyvisible for pollination. The female flower usually is hand-pollinatedimmediately after it is prepared; although a delay of several hours doesnot seem to reduce seed set. Pollen shed typically begins in the morningand may end when temperatures are above 30° C., or may begin later andcontinue throughout much of the day with more moderate temperatures.

Pollen is available from a flower with a recently opened corolla, butthe degree of corolla opening associated with pollen shed may varyduring the day. In many environments, it is possible to collect maleflowers and use them immediately without storage. In the southern U.S.and other humid climates, pollen shed occurs in the morning when femaleflowers are more immature and difficult to manipulate than in theafternoon, and the flowers may be damp from heavy dew. In thosecircumstances, male flowers are collected into envelopes or petri dishesin the morning and the open container is typically placed in adesiccator for about 4 h at a temperature of about 25° C. The desiccatormay be taken to the field in the afternoon and kept in the shade toprevent excessive temperatures from developing within it. Pollenviability can be maintained in flowers for up to 2 days when stored atabout 5° C. In a desiccator at 3° C., flowers can be stored successfullyfor several weeks; however, varieties may differ in the percentage ofpollen that germinates after long-term storage (Kuehl, 1961).

Either with or without emasculation of the female flower, handpollination can be carried out by removing the stamens and pistil with aforceps from a flower of the male parent and gently brushing the anthersagainst the stigma of the female flower. Access to the stamens can beachieved by removing the front sepal and keel petals, or piercing thekeel with closed forceps and allowing them to open to push the petalsaway. Brushing the anthers on the stigma causes them to rupture, and thehighest percentage of successful crosses is obtained when pollen isclearly visible on the stigma. Pollen shed can be checked by tapping theanthers before brushing the stigma. Several male flowers may have to beused to obtain suitable pollen shed when conditions are unfavorable, orthe same male may be used to pollinate several flowers with good pollenshed.

When male flowers do not have to be collected and dried in a desiccator,it may be desired to plant the parents of a cross adjacent to eachother. Plants usually are grown in rows 65 to 100 cm apart to facilitatemovement of personnel within the field nursery. Yield of self-pollinatedseed from an individual plant may range from a few seeds to more than1,000 as a function of plant density. A density of 30 plants/m of rowcan be used when 30 or fewer seeds per plant is adequate, 10 plants/mcan be used to obtain about 100 seeds/plant, and 3 plants/m usuallyresults in maximum seed production per plant. Densities of 12 plants/mor less commonly are used for artificial hybridization.

Multiple planting dates about 7 to 14 days apart usually are used tomatch parents of different flowering dates. When differences inflowering dates are extreme between parents, flowering of the laterparent can be hastened by creating an artificially short day orflowering of the earlier parent can be delayed by use of artificiallylong days or delayed planting. For example, crosses with genotypesadapted to the southern U.S. are made in northern U.S. locations bycovering the late genotype with a box, large can, or similar containerto create an artificially short photoperiod of about 12 h for about 15days beginning when there are three nodes with trifoliate leaves on themain stem. Plants induced to flower early tend to have flowers thatself-pollinate when they are small and can be difficult to prepare forhybridization.

Grafting can be used to hasten the flowering of late floweringgenotypes. A scion from a late genotype grafted on a stock that hasbegun to flower will begin to bloom up to 42 days earlier than normal(Kiihl et al., 1977). First flowers on the scion appear from 21 to 50days after the graft.

Observing pod development 7 days after pollination generally is adequateto identify a successful cross. Abortion of pods and seeds can occurseveral weeks after pollination, but the percentage of abortion usuallyis low if plant stress is minimized (Shibles et al., 1975). Pods thatdevelop from artificial hybridization can be distinguished fromself-pollinated pods by the presence of the calyx scar, caused byremoval of the sepals. The sepals begin to fall off as the pods mature;therefore, harvest should be completed at or immediately before the timethe pods reach their mature color. Harvesting pods early also avoids anyloss by shattering.

Once harvested, pods are typically air-dried at not more than 38° C.until the seeds contain 13% moisture or less, then the seeds are removedby hand. Seed can be stored satisfactorily at about 25° C. for up to ayear if relative humidity is 50% or less. In humid climates, germinationpercentage declines rapidly unless the seed is dried to 7% moisture andstored in an air-tight container at room temperature. Long-term storagein any climate is best accomplished by drying seed to 7% moisture andstoring it at 10° C. or less in a room maintained at 50% relativehumidity or in an air-tight container.

II. FURTHER EMBODIMENTS OF THE INVENTION

When the term soybean variety D4923560 is used in the context of thepresent invention, this also includes plants modified to include atleast a first desired heritable trait. Such plants may, in oneembodiment, be developed by a plant breeding technique calledbackcrossing, wherein essentially all of the desired morphological andphysiological characteristics of a variety are recovered in addition toa genetic locus transferred into the plant via the backcrossingtechnique. The term single locus converted plant as used herein refersto those soybean plants which are developed by a plant breedingtechnique called backcrossing, wherein essentially all of the desiredmorphological and physiological characteristics of a variety arerecovered in addition to the single locus transferred into the varietyvia the backcrossing technique. Backcrossing methods can be used withthe present invention to improve or introduce a characteristic into thepresent variety. The parental soybean plant which contributes the locusfor the desired characteristic is termed the nonrecurrent or donorparent. This terminology refers to the fact that the nonrecurrent parentis used one time in the backcross protocol and therefore does not recur.The parental soybean plant to which the locus or loci from thenonrecurrent parent are transferred is known as the recurrent parent asit is used for several rounds in the backcrossing protocol (Poehlman etal., 1995; Fehr, 1987a,b; Sprague and Dudley, 1988).

In a typical backcross protocol, the original variety of interest(recurrent parent) is crossed to a second variety (nonrecurrent parent)that carries the single locus of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a soybean plant isobtained wherein essentially all of the desired morphological andphysiological characteristics of the recurrent parent are recovered inthe converted plant, in addition to the single transferred locus fromthe nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single locus of the recurrent variety ismodified or substituted with the desired locus from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Soybean varieties can also be developed from more than two parents(Fehr, 1987a). The technique, known as modified backcrossing, usesdifferent recurrent parents during the backcrossing. Modifiedbackcrossing may be used to replace the original recurrent parent with avariety having certain more desirable characteristics or multipleparents may be used to obtain different desirable characteristics fromeach.

Many single locus traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single locus traits may or may not betransgenic; examples of these traits include, but are not limited to,male sterility, herbicide resistance, resistance to bacterial, fungal,or viral disease, insect resistance, restoration of male fertility,enhanced nutritional quality, yield stability, and yield enhancement.These comprise genes generally inherited through the nucleus.

Direct selection may be applied where the single locus acts as adominant trait. An example of a dominant trait is the herbicideresistance trait. For this selection process, the progeny of the initialcross are sprayed with the herbicide prior to the backcrossing. Thespraying eliminates any plants which do not have the desired herbicideresistance characteristic, and only those plants which have theherbicide resistance gene are used in the subsequent backcross. Thisprocess is then repeated for all additional backcross generations.

Selection of soybean plants for breeding is not necessarily dependent onthe phenotype of a plant and instead can be based on geneticinvestigations. For example, one may utilize a suitable genetic markerwhich is closely genetically linked to a trait of interest. One of thesemarkers may therefore be used to identify the presence or absence of atrait in the offspring of a particular cross, and hence may be used inselection of progeny for continued breeding. This technique may commonlybe referred to as marker assisted selection. Any other type of geneticmarker or other assay which is able to identify the relative presence orabsence of a trait of interest in a plant may also be useful forbreeding purposes. Procedures for marker assisted selection applicableto the breeding of soybeans are well known in the art. Such methods willbe of particular utility in the case of recessive traits and variablephenotypes, or where conventional assays may be more expensive, timeconsuming or otherwise disadvantageous. Types of genetic markers whichcould be used in accordance with the invention include, but are notnecessarily limited to, Simple Sequence Length Polymorphisms (SSLPs)(Williams et al., 1990), Randomly Amplified Polymorphic DNAs (RAPDs),DNA Amplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Arbitrary Primed Polymerase Chain Reaction (AP-PCR),Amplified Fragment Length Polymorphisms (AFLPs) (EP 534 858,specifically incorporated herein by reference in its entirety), andSingle Nucleotide Polymorphisms (SNPs) (Wang et al., 1998).

Many qualitative characters also have potential use as phenotype-basedgenetic markers in soybeans; however, some or many may not differ amongvarieties commonly used as parents (Bernard and Weiss, 1973). The mostwidely used genetic markers are flower color (purple dominant to white),pubescence color (brown dominant to gray), and pod color (brown dominantto tan). The association of purple hypocotyl color with purple flowersand green hypocotyl color with white flowers is commonly used toidentify hybrids in the seedling stage. Differences in maturity, height,hilum color, and pest resistance between parents can also be used toverify hybrid plants.

Many useful traits that can be introduced by backcrossing, as well asdirectly intro a plant, are those which are introduced by genetictransformation techniques. Genetic transformation may therefore be usedto insert a selected transgene into the soybean variety of the inventionor may, alternatively, be used for the preparation of transgenes whichcan be introduced by backcrossing. Methods for the transformation ofmany economically important plants, including soybeans, are well know tothose of skill in the art. Techniques which may be employed for thegenetic transformation of soybeans include, but are not limited to,electroporation, microprojectile bombardment, Agrobacterium-mediatedtransformation and direct DNA uptake by protoplasts.

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wound tissues ina controlled manner.

Protoplasts may also be employed for electroporation transformation ofplants (Bates, 1994; Lazzeri, 1995). For example, the generation oftransgenic soybean plants by electroporation of cotyledon-derivedprotoplasts was described by Dhir and Widholm in Intl. Patent Appl.Publ. No. WO 92/17598, the disclosure of which is specificallyincorporated herein by reference.

A particularly efficient method for delivering transforming DNA segmentsto plant cells is microprojectile bombardment. In this method, particlesare coated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. For the bombardment, cells in suspensionare concentrated on filters or solid culture medium. Alternatively,immature embryos or other target cells may be arranged on solid culturemedium. The cells to be bombarded are positioned at an appropriatedistance below the macroprojectile stopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System, whichcan be used to propel particles coated with DNA or cells through ascreen, such as a stainless steel or Nytex screen, onto a surfacecovered with target soybean cells. The screen disperses the particles sothat they are not delivered to the recipient cells in large aggregates.It is believed that a screen intervening between the projectileapparatus and the cells to be bombarded reduces the size of projectilesaggregate and may contribute to a higher frequency of transformation byreducing the damage inflicted on the recipient cells by projectiles thatare too large.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species. The application ofmicroprojectile bombardment for the transformation of soybeans isdescribed, for example, in U.S. Pat. No. 5,322,783, the disclosure ofwhich is specifically incorporated herein by reference in its entirety.

Agrobacterium-mediated transfer is another widely applicable system forintroducing gene loci into plant cells. An advantage of the technique isthat DNA can be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast. ModemAgrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations(Klee et al., 1985). Moreover, recent technological advances in vectorsfor Agrobacterium-mediated gene transfer have improved the arrangementof genes and restriction sites in the vectors to facilitate theconstruction of vectors capable of expressing various polypeptide codinggenes. The vectors described have convenient multi-linker regionsflanked by a promoter and a polyadenylation site for direct expressionof inserted polypeptide coding genes. Additionally, Agrobacteriumcontaining both armed and disarmed Ti genes can be used fortransformation.

In those plant strains where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene locus transfer. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (Fraley et al., 1985; U.S. Pat. No. 5,563,055). Use ofAgrobacterium in the context of soybean transformation has beendescribed, for example, by Chee and Slightom (1995) and in U.S. Pat. No.5,569,834, the disclosures of which are specifically incorporated hereinby reference in their entirety.

Transformation of plant protoplasts also can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., 1985; Omirulleh et al., 1993; Fromm et al., 1986;Uchimiya et al., 1986; Marcotte et al., 1988). The demonstrated abilityto regenerate soybean plants from protoplasts makes each of thesetechniques applicable to soybean (Dhir et al., 1991).

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a soybean plant according to theinvention. Non-limiting examples of particular genes and correspondingphenotypes one may choose to introduce into a soybean plant arepresented below.

A. Herbicide Resistance

Numerous herbicide resistance genes are known and may be employed withthe invention. An example is a gene conferring resistance to a herbicidethat inhibits the growing point or meristem, such as an imidazalinone ora sulfonylurea. Exemplary genes in this category code for mutant ALS andAHAS enzyme as described, for example, by Lee et al., (1988); Gleen etal., (1992) and Miki et al., (1990).

Resistance genes for glyphosate (resistance conferred by mutant5-enolpyruvl-3phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus phosphinothricin-acetyl transferase (bar) genes) may alsobe used. See, for example, U.S. Pat. No. 4,940,835 to Shah, et al.,which discloses the nucleotide sequence of a form of EPSPS which canconfer glyphosate resistance. Examples of specific EPSPS transformationevents conferring glyphosate resistance are provided by U.S. Pat. No.6,040,497.

A DNA molecule encoding a mutant aroA gene can be obtained under ATCCaccession number 39256, and the nucleotide sequence of the mutant geneis disclosed in U.S. Pat. No. 4,769,061 to Comai. European patentapplication No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a phosphinothricin-acetyltransferase gene isprovided in European application No. 0 242 246 to Leemans et al. DeGreefet al., (1989), describe the production of transgenic plants thatexpress chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy propionic acids and cycloshexones, such as sethoxydim andhaloxyfop are the Acct-S1, Accl-S2 and Acct-S3 genes described byMarshall et al., (1992).

Genes are also known conferring resistance to a herbicide that inhibitsphotosynthesis, such as a triazine (psbA and gs+ genes) and abenzonitrile (nitrilase gene). Przibila et al., (1991), describe thetransformation of Chlamydomonas with plasmids encoding mutant psbAgenes. Nucleotide sequences for nitrilase genes are disclosed in U.S.Pat. No. 4,810,648 to Stalker, and DNA molecules containing these genesare available under ATCC Accession Nos. 53435, 67441, and 67442. Cloningand expression of DNA coding for a glutathione S-transferase isdescribed by Hayes et al., (1992).

US Patent Application No: 20030135879 describes isolation of a gene fordicamba monooxygenase (DMO) from Pseudomonas maltophilia which isinvolved in the conversion of a herbicidal form of the herbicide dicambato a non-toxic 3,6-dichlorosalicylic acid and thus may be used forproducing plants tolerant to this herbicide.

B. Disease Resistance

Plant defenses are often activated by specific interaction between theproduct of a disease resistance gene (R) in the plant and the product ofa corresponding avirulence (Avr) gene in the pathogen. A plant line canbe transformed with cloned resistance gene to engineer plants that areresistant to specific pathogen strains. See, for example Jones et al.,(1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfulvum); Martin et al., (1993) (tomato Pto gene for resistance toPseudomonas syringae pv.); and Mindrinos et al., (1994) (ArabidopsisRSP2 gene for resistance to Pseudomonas syringae).

A viral-invasive protein or a complex toxin derived therefrom may alsobe used for viral disease resistance. For example, the accumulation ofviral coat proteins in transformed plant cells imparts resistance toviral infection and/or disease development effected by the virus fromwhich the coat protein gene is derived, as well as by related viruses.See Beachy et al., (1990). Coat protein-mediated resistance has beenconferred upon transformed plants against alfalfa mosaic virus, cucumbermosaic virus, tobacco streak virus, potato virus X, potato virus Y,tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.

A virus-specific antibody may also be used. See, for example,Tavladoraki et al., (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Logemann et al., (1992), for example, disclose transgenic plantsexpressing a barley ribosome-inactivating gene have an increasedresistance to fungal disease.

C. Insect Resistance

One example of an insect resistance gene includes a Bacillusthuringiensis protein, a derivative thereof or a synthetic polypeptidemodeled thereon. See, for example, Geiser et al., (1986), who disclosethe cloning and nucleotide sequence of a Bt δ-endotoxin gene. Moreover,DNA molecules encoding δ-endotoxin genes can be purchased from theAmerican Type Culture Collection, Manassas, Va., for example, under ATCCAccession Nos. 40098, 67136, 31995 and 31998. Another example is alectin. See, for example, Van Damme et al., (1994), who disclose thenucleotide sequences of several Clivia miniata mannose-binding lectingenes. A vitamin-binding protein may also be used, such as avidin. SeePCT application US93/06487, the contents of which are herebyincorporated by reference. This application teaches the use of avidinand avidin homologues as larvicides against insect pests.

Yet another insect resistance gene is an enzyme inhibitor, for example,a protease or proteinase inhibitor or an amylase inhibitor. See, forexample, Abe et al., (1987) (nucleotide sequence of rice cysteineproteinase inhibitor), Huub et al., (1993) (nucleotide sequence of cDNAencoding tobacco proteinase inhibitor I), and Sumitani et al., (1993)(nucleotide sequence of Streptomyces nitrosporeus α-amylase inhibitor).An insect-specific hormone or pheromone may also be used. See, forexample, the disclosure by Hammock et al., (1990), of baculovirusexpression of cloned juvenile hormone esterase, an inactivator ofjuvenile hormone.

Still other examples include an insect-specific antibody or animmunotoxin derived therefrom and a developmental-arrestive protein. SeeTaylor et al., (1994), who described enzymatic inactivation intransgenic tobacco via production of single-chain antibody fragments.

D. Male Sterility

Genetic male sterility is available in soybeans and can increase theefficiency with which hybrids are made, in that it can eliminate theneed to physically emasculate the soybean plant used as a female in agiven cross. (Brim and Stuber, 1973). Herbicide-inducible male sterilitysystems have also been described. (U.S. Pat. No. 6,762,344).

Where one desires to employ male-sterility systems, it may be beneficialto also utilize one or more male-fertility restorer genes. For example,where cytoplasmic male sterility (CMS) is used, hybrid seed productionrequires three inbred lines: (1) a cytoplasmically male-sterile linehaving a CMS cytoplasm; (2) a fertile inbred with normal cytoplasm,which is isogenic with the CMS line for nuclear genes (“maintainerline”); and (3) a distinct, fertile inbred with normal cytoplasm,carrying a fertility restoring gene (“restorer” line). The CMS line ispropagated by pollination with the maintainer line, with all of theprogeny being male sterile, as the CMS cytoplasm is derived from thefemale parent. These male sterile plants can then be efficientlyemployed as the female parent in hybrid crosses with the restorer line,without the need for physical emasculation of the male reproductiveparts of the female parent.

The presence of a male-fertility restorer gene results in the productionof fully fertile F₁ hybrid progeny. If no restorer gene is present inthe male parent, male-sterile hybrids are obtained. Such hybrids areuseful where the vegetative tissue of the soybean plant is utilized, butin many cases the seeds will be deemed the most valuable portion of thecrop, so fertility of the hybrids in these crops must be restored.Therefore, one aspect of the current invention concerns plants of thesoybean variety D4923560 comprising a genetic locus capable of restoringmale fertility in an otherwise male-sterile plant. Examples ofmale-sterility genes and corresponding restorers which could be employedwith the plants of the invention are well known to those of skill in theart of plant breeding (see, e.g., U.S. Pat. No. 5,530,191 and U.S. Pat.No. 5,684,242, the disclosures of which are each specificallyincorporated herein by reference in their entirety).

E. Modified Fatty Acid, Phytate and Carbohydrate Metabolism

Genes may be used conferring modified fatty acid metabolism. Forexample, stearyl-ACP desaturase genes may be used. See Knutzon et al.,(1992). Various fatty acid desaturases have also been described, such asa Saccharomyces cerevisiae OLE1 gene encoding Δ9-fatty acid desaturase,an enzyme which forms the monounsaturated palmitoleic (16:1) and oleic(18:1) fatty acids from palmitoyl (16:0) or stearoyl (18:0) CoA(McDonough et al., 1992); a gene encoding a stearoyl-acyl carrierprotein delta-9 desaturase from castor (Fox et al. 1993); Δ6- andΔ12-desaturases from the cyanobacteria Synechocystis responsible for theconversion of linoleic acid (18:2) to gamma-linolenic acid (18:3 gamma)(Reddy et al. 1993); a gene from Arabidopsis thaliana that encodes anomega-3 desaturase (Arondel et al. 1992)); plant Δ9-desaturases (PCTApplication Publ. No. WO 91/13972) and soybean and Brassica Δ15desaturases (European Patent Application Publ. No. EP 0616644).

Phytate metabolism may also be modified by introduction of aphytase-encoding gene to enhance breakdown of phytate, adding more freephosphate to the transformed plant. For example, see Van Hartingsveldtet al., (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. In soybean, this, for example, could beaccomplished by cloning and then reintroducing DNA associated with thesingle allele which is responsible for soybean mutants characterized bylow levels of phytic acid. See Raboy et al., (2000).

A number of genes are known that may be used to alter carbohydratemetabolism. For example, plants may be transformed with a gene codingfor an enzyme that alters the branching pattern of starch. See Shirozaet al., (1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al., (1992)(production of transgenic plants that express Bacillus licheniformisα-amylase), Elliot et al., (1993) (nucleotide sequences of tomatoinvertase genes), Sergaard et al., (1993) (site-directed mutagenesis ofbarley α-amylase gene), and Fisher et al., (1993) (maize endospermstarch branching enzyme II). The Z10 gene encoding a 10 kD zein storageprotein from maize may also be used to alter the quantities of 10 kDZein in the cells relative to other components (Kirihara et al., 1988).

III. ORIGIN AND BREEDING HISTORY OF AN EXEMPLARY SINGLE LOCUS CONVERTEDPLANT

It is known to those of skill in the art that, by way of the techniqueof backcrossing, one or more traits may be introduced into a givenvariety while otherwise retaining essentially all of the traits of thatvariety. An example of such backcrossing to introduce a trait into astarting variety is described in U.S. Pat. No. 6,140,556, the entiredisclosure of which is specifically incorporated herein by reference.The procedure described in U.S. Pat. No. 6,140,556 can be summarized asfollows: The soybean variety known as Williams '82 [Glycine max L.Merr.] (Reg. No. 222, PI 518671) was developed using backcrossingtechniques to transfer a locus comprising the Rps₁ gene to the varietyWilliams (Bernard and Cremeens, 1988). Williams '82 is a composite offour resistant lines from the BC₆F₃ generation, which were selected from12 field-tested resistant lines from Williams x Kingwa. The varietyWilliams was used as the recurrent parent in the backcross and thevariety Kingwa was used as the source of the Rps₁ locus. This gene locusconfers resistance to 19 of the 24 races of the fungal agent phytopthorarot.

The F₁ or F₂ seedlings from each backcross round were tested forresistance to the fungus by hypocotyl inoculation using the inoculum ofrace 5. The final generation was tested using inoculum of races 1 to 9.In a backcross such as this, where the desired characteristic beingtransferred to the recurrent parent is controlled by a major gene whichcan be readily evaluated during the backcrossing, it is common toconduct enough backcrosses to avoid testing individual progeny forspecific traits such as yield in extensive replicated tests. In general,four or more backcrosses are used when there is no evaluation of theprogeny for specific traits, such as yield. As in this example, lineswith the phenotype of the recurrent parent may be composited without theusual replicated tests for traits such as yield, protein or oilpercentage in the individual lines.

The variety Williams '82 is comparable to the recurrent parent varietyWilliams in all traits except resistance to phytopthora rot. Forexample, both varieties have a relative maturity of 38, indeterminatestems, white flowers, brown pubescence, tan pods at maturity and shinyyellow seeds with black to light black hila.

IV. TISSUE CULTURES AND IN VITRO REGENERATION OF SOYBEAN PLANTS

A further aspect of the invention relates to tissue cultures of thesoybean variety designated D4923560. As used herein, the term “tissueculture” indicates a composition comprising isolated cells of the sameor a different type or a collection of such cells organized into partsof a plant. Exemplary types of tissue cultures are protoplasts, calliand plant cells that are intact in plants or parts of plants, such asembryos, pollen, flowers, leaves, roots, root tips, anthers, and thelike. In a preferred embodiment, the tissue culture comprises embryos,protoplasts, meristematic cells, pollen, leaves or anthers.

Exemplary procedures for preparing tissue cultures of regenerablesoybean cells and regenerating soybean plants therefrom, are disclosedin U.S. Pat. No. 4,992,375; U.S. Pat. No. 5,015,580; U.S. Pat. No.5,024,944, and U.S. Pat. No. 5,416,011, each of the disclosures of whichis specifically incorporated herein by reference in its entirety.

An important ability of a tissue culture is the capability to regeneratefertile plants. This allows, for example, transformation of the tissueculture cells followed by regeneration of transgenic plants. Fortransformation to be efficient and successful, DNA must be introducedinto cells that give rise to plants or germ-line tissue.

Soybeans typically are regenerated via two distinct processes; shootmorphogenesis and somatic embryogenesis (Finer, 1996). Shootmorphogenesis is the process of shoot meristem organization anddevelopment. Shoots grow out from a source tissue and are excised androoted to obtain an intact plant. During somatic embryogenesis, anembryo (similar to the zygotic embryo), containing both shoot and rootaxes, is formed from somatic plant tissue. An intact plant rather than arooted shoot results from the germination of the somatic embryo.

Shoot morphogenesis and somatic embryogenesis are different processesand the specific route of regeneration is primarily dependent on theexplant source and media used for tissue culture manipulations. Whilethe systems are different, both systems show variety-specific responseswhere some lines are more responsive to tissue culture manipulationsthan others. A line that is highly responsive in shoot morphogenesis maynot generate many somatic embryos. Lines that produce large numbers ofembryos during an ‘induction’ step may not give rise to rapidly-growingproliferative cultures. Therefore, it may be desired to optimize tissueculture conditions for each soybean line. These optimizations mayreadily be carried out by one of skill in the art of tissue culturethrough small-scale culture studies. In addition to line-specificresponses, proliferative cultures can be observed with both shootmorphogenesis and somatic embryogenesis. Proliferation is beneficial forboth systems, as it allows a single, transformed cell to multiply to thepoint that it will contribute to germ-line tissue.

Shoot morphogenesis was first reported by Wright et al. (1986) as asystem whereby shoots were obtained de novo from cotyledonary nodes ofsoybean seedlings. The shoot meristems were formed subepidermally andmorphogenic tissue could proliferate on a medium containing benzyladenine (BA). This system can be used for transformation if thesubepidermal, multicellular origin of the shoots is recognized andproliferative cultures are utilized. The idea is to target tissue thatwill give rise to new shoots and proliferate those cells within themeristematic tissue to lessen problems associated with chimerism.Formation of chimeras, resulting from transformation of only a singlecell in a meristem, are problematic if the transformed cell is notadequately proliferated and does not does not give rise to germ-linetissue. Once the system is well understood and reproducedsatisfactorily, it can be used as one target tissue for soybeantransformation.

Somatic embryogenesis in soybean was first reported by Christianson etal. (1983) as a system in which embryogenic tissue was initiallyobtained from the zygotic embryo axis. These embryogenic cultures wereproliferative but the repeatability of the system was low and the originof the embryos was not reported. Later histological studies of adifferent proliferative embryogenic soybean culture showed thatproliferative embryos were of apical or surface origin with a smallnumber of cells contributing to embryo formation. The origin of primaryembryos (the first embryos derived from the initial explant) isdependent on the explant tissue and the auxin levels in the inductionmedium (Hartweck et al., 1988). With proliferative embryonic cultures,single cells or small groups of surface cells of the ‘older’ somaticembryos form the ‘newer’ embryos.

Embryogenic cultures can also be used successfully for regeneration,including regeneration of transgenic plants, if the origin of theembryos is recognized and the biological limitations of proliferativeembryogenic cultures are understood. Biological limitations include thedifficulty in developing proliferative embryogenic cultures and reducedfertility problems (culture-induced variation) associated with plantsregenerated from long-term proliferative embryogenic cultures. Some ofthese problems are accentuated in prolonged cultures. The use of morerecently cultured cells may decrease or eliminate such problems.

VI. DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided:

A: When used in conjunction with the word “comprising” or other openlanguage in the claims, the words “a” and “an” denote “one or more.”

Allele: Any of one or more alternative forms of a gene locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny, for example a first generation hybrid (F₁), back to one of theparents of the hybrid progeny. Backcrossing can be used to introduce oneor more single locus conversions from one genetic background intoanother.

Brown Stem Rot: This is a visual disease score from 1 to 9 comparing allgenotypes in a given test. The score is based on leaf symptoms ofyellowing and necrosis caused by brown stem rot. A score of 1 indicatesno symptoms. Visual scores range to a score of 9 which indicates severesymptoms of leaf yellowing and necrosis.

Chromatography: A technique wherein a mixture of dissolved substancesare bound to a solid support followed by passing a column of fluidacross the solid support and varying the composition of the fluid. Thecomponents of the mixture are separated by selective elution.

Crossing: The mating of two parent plants.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a cytoplasmic or nuclear genetic factor conferring malesterility or a chemical agent.

Emergence: This is a score indicating the ability of a seed to emergefrom the soil after planting. Each genotype is given a 1 to 9 scorebased on its percent of emergence. A score of 1 indicates an excellentrate and percent of emergence, an intermediate score of 5 indicatesaverage ratings and a 9 score indicates a very poor rate and percent ofemergence.

Enzymes: Molecules which can act as catalysts in biological reactions.

F₁ Hybrid: The first generation progeny of the cross of two nonisogenicplants.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Iron-Deficiency Chlorosis: A plant scoring system ranging from 1 to 9based on visual observations. A score of 1 means no stunting of theplants or yellowing of the leaves and a score of 9 indicates the plantsare dead or dying caused by iron-deficiency chlorosis, a score of 5means plants have intermediate health with some leaf yellowing.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Lodging Resistance: Lodging is rated on a scale of 1 to 9. A score of 1indicates erect plants. A score of 5 indicates plants are leaning at a45 degree(s) angle in relation to the ground and a score of 9 indicatesplants are laying on the ground.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Maturity Date: Plants are considered mature when 95% of the pods havereached their mature color. The maturity date is typically described inmeasured days after August 31 in the northern hemisphere.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Phytophthora Tolerance: Tolerance to Phytophthora root rot is rated on ascale of 1 to 9, with a score of 1 being the best or highest toleranceranging down to a score of 9, which indicates the plants have notolerance to Phytophthora.

Plant Height: Plant height is taken from the top of soil to the top nodeof the plant and is measured in inches.

Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Regeneration: The development of a plant from tissue culture.

Relative Maturity: The maturity grouping designated by the soybeanindustry over a given growing area. This figure is generally dividedinto tenths of a relative maturity group. Within narrow comparisons, thedifference of a tenth of a relative maturity group equates very roughlyto a day difference in maturity at harvest.

Seed Protein Peroxidase Activity. Seed protein peroxidase activity isdefined as a chemical taxonomic technique to separate varieties based onthe presence or absence of the peroxidase enzyme in the seed coat. Thereare two types of soybean varieties, those having high peroxidaseactivity (dark red color) and those having low peroxidase activity (nocolor).

Seed Yield (Bushels/Acre): The yield in bushels/acre is the actual yieldof the grain at harvest.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Shattering: The amount of pod dehiscence prior to harvest. Poddehiscence involves seeds falling from the pods to the soil. This is avisual score from 1 to 9 comparing all genotypes within a given test. Ascore of 1 means pods have not opened and no seeds have fallen out. Ascore of 5 indicates approximately 50% of the pods have opened, withseeds falling to the ground and a score of 9 indicates 100% of the podsare opened.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing, wherein essentially allof the desired morphological and physiological characteristics of asoybean variety are recovered in addition to the characteristics of thesingle locus transferred into the variety via the backcrossing techniqueand/or by genetic transformation.

Substantially Equivalent: A characteristic that, when compared, does notshow a statistically significant difference (e.g., p=0.05) from themean.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic locus comprising a sequence which has beenintroduced into the genome of a soybean plant by transformation.

VII. DEPOSIT INFORMATION

A deposit of the soybean variety D4923560, disclosed above and recitedin the claims, has been made with the American Type Culture Collection(ATCC), 10801 University Blvd., Manassas, Va. 20110-2209. The date ofdeposit was ______. All restrictions upon the deposit have been removed,and the deposit is intended to meet all of the requirements of 37 C.F.R.§1.801-1.809. The accession number for those deposited seeds of soybeanvariety D4923560 is ATCC Accession No. ______. The deposit will bemaintained in the depository for a period of 30 years, or 5 years afterthe last request, or for the effective life of the patent, whichever islonger, and will be replaced if necessary during that period.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A seed of soybean variety D4923560 (ATCC Accession No. ______).
 2. Aplant of soybean variety D4923560 (ATCC Accession No. ______).
 3. Aplant part of the plant of claim
 2. 4. The plant part of claim 3,further defined as pollen, an ovule or a cell.
 5. A tissue culture ofregenerable cells of soybean variety D4923560 (ATCC Accession No.______).
 6. The tissue culture of claim 5, wherein the regenerable cellsare from embryos, meristematic cells, pollen, leaves, roots, root tips,anther, pistil, flower, seed, boll or stem.
 7. A soybean plantregenerated from the tissue culture of claim 5, wherein the regeneratedsoybean plant expresses all of the physiological and morphologicalcharacteristics of the soybean variety D4923560 (ATCC Accession No.______).
 8. A method of producing soybean seed, comprising crossing aplant of soybean variety D4923560 (ATCC Accession No. ______) withitself or a second soybean plant.
 9. The method of claim 8, furtherdefined as a method of preparing hybrid soybean seed, comprisingcrossing a plant of soybean variety D4923560 (ATCC Accession No. ______)a second, distinct soybean plant.
 10. An F₁ hybrid seed produced by themethod of claim
 9. 11. A method of producing a plant of soybean varietyD4923560 (ATCC Accession No. ______) comprising an added desired trait,the method comprising introducing a transgene conferring the desiredtrait into a plant of soybean variety D4923560.
 12. The method of claim11, wherein the desired trait is selected from the group consisting ofmale sterility, herbicide tolerance, insect or pest resistance, diseaseresistance, modified fatty acid metabolism, modified carbohydratemetabolism and modified soybean fiber characteristics.
 13. The method ofclaim 12, wherein the desired trait is herbicide tolerance and thetolerance is conferred to an herbicide selected from the groupconsisting of glyphosate, sulfonylurea, imidazalinone, dicamba,glufosinate, phenoxy proprionic acid, cycloshexone, triazine,benzonitrile and broxynil.
 14. The method of claim 11, wherein thedesired trait is insect resistance and the transgene encodes a Bacillusthuringiensis (Bt) endotoxin.
 15. A plant produced by the method ofclaim
 11. 16. A method of introducing a single locus conversion intosoybean variety D4923560 comprising: (a) crossing a plant of varietyD4923560 (ATCC Accession No. ______) with a second plant comprising adesired single locus to produce F1 progeny plants; (b) selecting F1progeny plants that have the single locus to produce selected F1 progenyplants; (c) crossing the selected progeny plants with at least a firstplant of variety D4923560 to produce backcross progeny plants; (d)selecting backcross progeny plants that have the single locus andphysiological and morphological characteristics of soybean varietyD4923560 to produce selected backcross progeny plants; and (e) repeatingsteps (c) and (d) one or more times in succession to produce selectedsecond or higher backcross progeny plants that comprise the single locusand the physiological and morphological characteristics of soybeanvariety D4923560 when grown in the same environmental conditions. 17.The method of claim 16, wherein the single locus confers a traitselected from the group consisting of male sterility; herbicidetolerance; insect or pest resistance; disease resistance; modified fattyacid metabolism; modified carbohydrate metabolism; and modified soybeanfiber characteristics.
 18. The method of claim 17, wherein the trait istolerance to an herbicide selected from the group consisting ofglyphosate, sulfonylurea, imidazalinone, dicamba, glufosinate, phenoxyproprionic acid, cycloshexone, triazine, benzonitrile and broxynil. 19.The method of claim 17, wherein the trait is insect resistance and theinsect resistance is conferred by a transgene encoding a Bacillusthuringiensis endotoxin.
 20. A plant of soybean variety D4923560 (ATCCAccession No. ______), further defined as comprising a single locusconversion.
 21. A method of producing an inbred soybean plant derivedfrom the soybean variety D4923560, the method comprising the steps of:(a) preparing a progeny plant derived from soybean variety D4923560(ATCC Accession No. ______) by crossing a plant of the soybean varietyD4923560 with a soybean plant of a second variety; (b) crossing theprogeny plant with itself or a second plant to produce a seed of aprogeny plant of a subsequent generation; (c) growing a progeny plant ofa subsequent generation from said seed and crossing the progeny plant ofa subsequent generation with itself or a second plant; and (d) repeatingsteps (b) and (c) for an additional 3-10 generations with sufficientinbreeding to produce an inbred soybean plant derived from the soybeanvariety D4923560.
 22. A method of producing a commodity plant productcomprising obtaining the plant of claim 2 or a part thereof andproducing said commodity plant product therefrom.
 23. The method ofclaim 22, wherein the commodity plant product is protein concentrate,protein isolate, soybean hulls, meal, flour or oil.